Combined thermal devices for thermal cycling

ABSTRACT

The invention relates to systems and methods including a combination of thermal generating device technologies to achieve more efficiency and accuracy in PCR temperature cycling of nucleic samples undergoing amplification.

This application is a continuation of patent application Ser. No.12/966,583 filed Dec. 13, 2010, now U.S. Pat. No. 8,507,257, which is adivisional of patent application Ser. No. 11/771,067, filed Jun. 29,2007, now U.S. Pat. No. 7,851,185, which claims the benefit ofProvisional Patent Application No. 60/806,440, filed on Jun. 30, 2006,which is incorporated herein by this reference.

BACKGROUND

1. Field of the Invention

The present invention relates to systems and methods for efficientthermal cycling in DNA amplification using a combination of energysources, including electrical and/or magnetic (hereafterelectromagnetic) radiation as an energy source.

2. Discussion of the Background

The detection of nucleic acids is central to medicine, forensic science,industrial processing, crop and animal breeding, and many other fields.The ability to detect disease conditions (e.g., cancer), infectiousorganisms (e.g., HIV), genetic lineage, genetic markers, and the like,is ubiquitous technology for disease diagnosis and prognosis, markerassisted selection, identification of crime scene features, the abilityto propagate industrial organisms and many other techniques.Determination of the integrity of a nucleic acid of interest can berelevant to the pathology of an infection or cancer.

One of the most powerful and basic technologies to detect smallquantities of nucleic acids is to replicate some or all of a nucleicacid sequence many times, and then analyze the amplification products.Polymerase chain reaction (PCR) is a well-known technique for amplifyingDNA. With PCR, one can produce millions of copies of DNA starting from asingle template DNA molecule. PCR includes phases of “denaturation,”“annealing,” and “extension.” These phases are part of a cycle which isrepeated a number of times so that at the end of the process there areenough copies to be detected and analyzed. For general detailsconcerning PCR, see Sambrook and Russell, Molecular Cloning—A LaboratoryManual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2005) and PCR Protocols A Guide to Methods and Applications, M.A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).

The PCR process phases of denaturing, annealing, and extension occur atdifferent temperatures and cause target DNA molecule samples toreplicate themselves. Temperature cycling (thermocyling) requirementsvary with particular nucleic acid samples and assays. In the denaturingphase, a double stranded DNA (dsDNA) is thermally separated into singlestranded DNA (ssDNA). During the annealing phase, primers are attachedto the single stand DNA molecules. Single strand DNA molecules grow todouble stranded DNA again in the extension phase through specificbindings between nucleotides in the PCR solution and the single strandDNA. Typical temperatures are 95° C. for denaturing, 55° C. forannealing, and 72° C. for extension. The temperature is held at eachphase for a certain amount of time which may be a fraction of a secondup to a few tens of seconds. The DNA is doubled at each cycle; itgenerally takes 20 to 40 cycles to produce enough DNA for theapplications. To have good yield of target product, one has toaccurately control the sample temperatures at the different phases to aspecified degree.

More recently, a number of high throughput approaches to performing PCRand other amplification reactions have been developed, e.g., involvingamplification reactions in microfluidic devices, as well as methods fordetecting and analyzing amplified nucleic acids in or on the devices.Thermal cycling of the sample for amplification is usually accomplishedin one of two methods. In the first method, the sample solution isloaded into the device and the temperature is cycled in time, much likea conventional PCR instrument. In the second method, the sample solutionis pumped continuously through spatially varying temperature zones. See,for example, Lagally et al. (Analytical Chemistry 73:565-570 (2001)),Kopp et al. (Science 280:1046-1048 (1998)), Park et al. (AnalyticalChemistry 75:6029-6033 (2003)), Hahn et al. (WO 2005/075683),Enzelberger et al. (U.S. Pat. No. 6,960,437) and Knapp et al. (U.S.Patent Application Publication No. 2005/0042639).

Many detection methods require a determined large number of copies(millions, for example) of the original DNA molecule, in order for theDNA to be characterized. Because the total number of cycles is fixedwith respect to the number of desired copies, the only way to reduce theprocess time is to reduce the length of a cycle. Thus, the total processtime may be significantly reduced by rapidly heating and cooling samplesto process phase temperatures while accurately maintaining thosetemperatures for the process phase duration.

Accordingly, what is desired is a system and method for rapidly andaccurately changing process temperatures in PCR processes.

SUMMARY OF THE INVENTION

The present invention relates to systems and methods for rapidtemperature change in microfluidic thermal cycling.

In one aspect, the present invention provides a method for cycling thetemperature of a nucleic acid sample. In one embodiment, the methodincludes: (a) controlling a heating device to cause a temperature of thesample to be at or about a first desired temperature for at least afirst time period; (b) after expiration of the first time period,increasing the output of an electromagnetic heating source to cause thetemperature of the sample to be at or about a second desired temperaturefor at least a second time period; (c) during said second time period,lowering the amount of heat the heating device provides to the sample;and (d) immediately after expiration of the second time period, loweringthe output of the electromagnetic heating source and controlling theheating device to cause the temperature of the sample to be at or abouta third desired temperature for a third time period, wherein the firsttemperature is less than the second temperature and the thirdtemperature is less than the first temperature. In some embodiments,steps (a) through (d) occur while the sample is flowing through achannel (e.g., a microfluidic channel).

In another embodiment, the method includes: heating the sample to afirst temperature for a first time period using a thermoelectric device;heating the sample to a second temperature for a second time periodusing primarily an electromagnetic heat source; cooling the sample to athird temperature; and maintaining the third temperature for a thirdtime period using the thermoelectric device, wherein the secondtemperature is higher than the first and the first temperature is higherthan the third.

In another embodiment, the method includes: (a) heating the nucleic acidsample to about a first temperature; (b) after heating the sample toabout the first temperature, maintaining the temperature of the sampleat about the first temperature for a first period of time; (c) afterexpiration of the first period of time, heating the sample to about asecond temperature; (d) after heating the sample to the secondtemperature, maintaining the temperature of the sample at about thesecond temperature for a second period of time; (e) after expiration ofthe second period of time, cooling the sample to about a thirdtemperature; and (f) after cooling the sample to the third temperature,maintaining the temperature of the sample at about the third temperaturefor a third period of time, wherein the first temperature is less thanthe second temperature and greater than the third temperature, and thestep of heating the sample to the second temperature consists primarilyof using one or more non-contact heating elements to heat the sample tothe second temperature.

In another aspect, the present invention provides a system for cyclingthe temperature of a nucleic acid sample. In one embodiment, the systemincludes: a nucleic acid sample container operable to receive a nucleicacid sample; a first heating device; and a second heating device,wherein the first heating device is configured to heat the nucleic acidsample to at least about a first temperature, the second heating deviceis configured to heat the nucleic acid sample to a second and thirdtemperature, the first temperature is associated with a denaturing phaseof a PCR process, the first heating device is a non-contact heatingdevice, and the second heating device is a contact heating device.

The above and other embodiments of the present invention are describedbelow with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements. Additionally, the left-most digit(s) of areference number identifies the drawing in which the reference numberfirst appears.

FIG. 1 depicts an apparatus in accordance with an exemplary embodimentof the invention.

FIG. 2 depicts an exemplary desired PCR temperature cycle.

FIG. 3 depicts a temperature characteristic of a heating device and ofan electromagnetic heating device.

FIGS. 4 and 5 depict a temperature cycle.

FIG. 6 depicts steps in an exemplary method in accordance with anembodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

With reference to FIG. 1, an exemplary embodiment of an apparatus 100relating to the present invention may include: a microfluidic device 101or other device for containing a sample containing a nucleic acid andPCR reagents (which PCR reagents may include PCR primers, dNTPs,polymerase enzymes, salts, buffers, surface-passivating agents, and thelike), a heat spreader 102, a heating device 103, a heat sink 104, anelectromagnetic heat source 105, a contact temperature sensing device107, a non-contact temperature sensing device 108, a controller 109 anda fan 110.

In some embodiments, device 101 includes a microfluidic channelconfigured to receive the sample. The sample may flow through thechannel as its temperature is cycled, as described herein. Moving thesample through the microfluidic channel can be accomplished by a varietyof methods, for example, via conventional methods of pressure-drivenflow (e.g., using a pump to create a pressure differential) and the flowrates can vary, for example between 10 nanoliters per minute to 1 ml perminute.

In the embodiment illustrated, heating device 103 is thermally incontact with the microfluidic device 101 through the metal heaterspreader 102. Device 103 may be implemented using a thermoelectriccooler (TEC) (also referred to as a Peltier device), a resistive heater,a chemical heater, or other heating device. A suitable TEC may beavailable from Melcor Corporation of Trenton, N.J. (see part numberHT6-6-21X43). In some embodiments, heating device 103 may have adifferent temperature resolution than heat source 105. Morespecifically, in some embodiments, heating device 103 may have a finertemperature resolution than heat source 105.

As mentioned above, device 103 may be implemented using a Peltierdevice, a widely used component in laboratory instrumentation andequipment, well known among those familiar with such equipment, andreadily available from commercial suppliers such as Melcor. A Peltierdevice is a solid-state device that can function as a heat pump, suchthat when an electric current flows through two dissimilar conductors,the junction of the two conductors will either absorb or release heatdepending on the direction of current flow. A typical Peltier deviceconsists of two ceramic or metallic plates separated by a semiconductormaterial, such as bismuth telluride. The direction of heat flow is afunction of the direction of the electric current and the nature of thecharge carrier in the semiconductor (i.e., n-type or p-type). Peltierdevices can be arranged and/or electrically connected in an apparatus ofthe present invention to heat or to cool a PCR process taking place inmicrofluidic device 101.

The size of heat spreader 102 is related to the sizes of the heatingdevice 103 and microfluidic device 101. In an exemplary embodiment, aheat spreader of 20 mm×40 mm is used. Heat sink 104 removes waste heatfrom heating device 103. The assembly comprising the heat sink 104,heating device 103, and heater spreader 102 may be screwed together,bonded together, or clamped in place. Thermal coupling may be enhancedby the use of thermally conductive adhesives, greases, pastes,thermoconducting pads (e.g., a SIL-PAD product available from theBerquist Company of Chanhassen, Minn.).

A switchable fan 110 may be used to increase airflow towards theassembly and/or heat sink 104 in particular. The fan 110 and heat sink104 can function together to quickly remove heat from the assembly. Insome embodiments, fan 110 may be left on continuously during for anentire PCR process.

Electromagnetic heat source 105 may radiate energy 106 directed towardthe microfluidic device 101 surface. A suitable electromagnetic heatsource is any device which generates an electric and/or magnetic fieldwhich may be used to heat microfluidic device 101. An exemplaryelectromagnetic heat source 105 may be an infrared source including atungsten filament bulb, such as one from the GE XR series, or a laser.Heat source 105 may be located ½ of an inch or less to twelve inches ormore from the surface of microfluidic device 101. Preferably, heatsource 105 is located at an angle so as to facilitate real-time PCRmonitoring of the microfluidic zone and is located between about 2-6inches from the surface of device 101.

Contact temperature sensing device 107 may be located inside or on themicrofluidic device 101 surface. Suitable temperature sensors include athin film wire, embedded wire, thermocouple, RTD, resistor, or solidstate device. In some embodiments, a temperature measuring sensoravailable from Analog Devices is used to implement device 107 (e.g., theAnalog Devices AD590 temperature transducer may be used). In someembodiments, a non-contact temperature sensing device 108, such as apyrometer manufactured by Mikron, may be used in addition to or insteadof contact temperature sensing device 107.

Controller 109 may be used to energize and deenergize heating device 103and electromagnetic heat source 105 in a thermostatic fashion such thatthe temperature sensed by sensor 107 and/or 108 (e.g., the temperatureof a region of microfluidic device 101) is at, or approximately at, adesired temperature for a desired period of time. Controller 109 includeone or more computers or other programmable devices which may beprogrammed to control heating device 103, electromagnetic heat source105, and/or fan 111 in response to the expiration of a timer andtemperature measurements from contact temperature sensor 107 and/ornon-contact temperature sensor 108.

FIG. 2 depicts an exemplary desired PCR cycle. In this exemplary cycle,phase 211 is the extension phase, phase 212 is the denaturation phase,and phase 213 is the annealing phase. Typically, the denaturation phase212 requires less precision of temperature and time control, providedthat a minimum temperature point is achieved homogeneously in themicrofluidic device 101.

In a preferred embodiment, controller 109 is programmed to use primarilyheating device 103 to heat and/or cool microfluidic device 101 duringextension phase 211 and annealing phase 213. That is, in someembodiments, source 105 may be turned “off” during the extension phaseand annealing phase or may output a lower level of radiation 106 duringthese phases than it outputs during denaturation phase 212. Controller109 is operable to energize the heating and/or cooling ability ofheating device 103 such that the desired temperatures are quicklyreached and maintained for the desired times. For example, an extensionphase 211 may have a duration of about 5 seconds and a desiredtemperature of about 72° C. An exemplary annealing phase 213 may have adesired duration of 2 seconds and a desired temperature of about 55° C.

In the preferred embodiment, electromagnetic heat source 105 provides tomicrofluidic device 101 a greater amount of heat during the denaturationphase 212 than during the other two phases of the PCR cycle.Additionally, during denaturation phase 212, device 103 may be operatedto provide less heat to device 101 than device 103 is configured toprovide to device 101 during the other two phases of the PCR cycle.Accordingly, in some embodiments, device 103 may actually draw heat fromdevice 101 during denaturation phase 212. An exemplary desireddenaturation phase 212 may last about 500 ms at a desired temperature ofabout 95° C.

FIG. 3 depicts exemplary plots of a heating device 103 temperaturecharacteristic 301 and source 105 temperature characteristic 303.Characteristic 301 illustrates that the heating device 103 is controlledby the controller 109 such that the desired temperature control of thesample for the extension phase 211 and annealing phase 213 issubstantially provided through the functioning of the heating device103. Characteristic 303 illustrates that the electromagnetic heat source105 is controlled by the controller 109 such that the desiredtemperature control of the sample for denaturation phase 212 issubstantially provided through the functioning of the electromagneticheat source 105.

FIGS. 4 and 5 depict time and temperature graphs for a sample containedin device 101 when heat sources 103 and 105 are operated according tothe diagram shown in FIG. 3. Shaded area 441 represents the part of thecycle for which device 103 provides thermal input. Period A, whichoccurs in area 440, represents the duration of an energy input fromsource 105.

Accordingly, and with reference to FIG. 5, fast thermal ramp/rise ratesmay be achieved as indicated by slope 530. Furthermore, high coolingrates may be achieved as indicated by slope 531.

Referring now to FIG. 6, FIG. 6 is a flow chart illustrating a process,according to some embodiments of the invention, for cycling thetemperature of a sample that is present in device 101.

The process may begin in step S601, where the controller controlsthermostatically heating device 103 such that the extension phasetemperature is reached and maintained for a desired duration of theextension phase 211. While step S601 is being performed, source 105 maybe in an “off” state.

In step S603, which preferably does not occur until about immediatelyafter the expiration of the desired extension phase duration, controller109 increases the output of electromagnetic heat source 105. Preferably,the output of source 105 is raised to a level that causes thetemperature of the sample to rapidly increase to the denaturation phasetemperature.

In step 605, controller 109 may control thermostatically one or more ofthe heating and cooling devices of apparatus 100 so that the sample iskept at the denaturation phase temperature for the desired duration ofthe denaturation phase 212.

At or about the same time controller 109 causes heat source 105 to heatthe sample to the denature temperature, controller 109 may controldevice 103 such that the heat provided by device 103 to device 101 isless than the heat device 103 provided to device 101 during step S601(i.e., during extension phase 211).

In step S607, which preferably does not occur until about immediatelyafter the expiration of the desired denature phase duration, controller109 lowers the energy output (e.g., turns off) electromagnetic heatsource 105 and controls heating device 103 such that the annealing phasetemperature is reached. In some embodiments, step S607 includes usingthe cooling capability of the heating device to quickly drive thetemperature of the sample down from the denaturation phase temperatureto the annealing phase temperature as shown in ramp 531. In someembodiments, step S607 also includes activating and directing fan 110 atthe microfluidic device 101 and/or heat sink 104 to drive thetemperature down rapidly as shown in ramp 631.

In step S609, controller 109 may control thermostatically one or more ofthe heating and cooling devices of apparatus 100 so that the sample iskept at the annealing phase temperature for the desired duration of theannealing phase 213. For example, if a temperature sensor (e.g., sensor107 or 108) indicates that the temperature of the sample is too low,then controller 109 may control a heat source (e.g., device 103 orsource 105) to add more heat to the sample, and if a temperature sensor108 indicates that the temperature of the sample is too low, thencontroller 109 may control device 103 so that it draws heat from thesample.

Embodiments of the present invention have been fully described abovewith reference to the drawing figures. Although the invention has beendescribed based upon these preferred embodiments, it would be apparentto those of skill in the art that certain modifications, variations, andalternative constructions could be made to the described embodimentswithin the spirit and scope of the invention.

Furthermore, one of skill in the art will recognize that temperaturesenumerated in the following claims should be interpreted to mean “at orabout” the enumerated temperature.

For the claims below the words “a” and “an” should be construed as “oneor more.”

What is claimed is:
 1. A method for heating a nucleic acid sample in amicrofluidic device by using a heating device and an electromagneticheating device, the method comprising: (a) controlling the heatingdevice to cause a temperature of the sample to be at or about a firstdesired temperature for at least a first time period; (b) afterexpiration of the first time period, increasing the output of theelectromagnetic heating source to cause the temperature of the sample tobe at or about a second desired temperature for at least a second timeperiod; (c) during said second time period, lowering the amount of heatthe heating device provides to the sample; and (d) immediately afterexpiration of the second time period, lowering the output of theelectromagnetic heating source and controlling the heating device tocause the temperature of the sample to be at or about a third desiredtemperature for a third time period, wherein the first temperature isless than the second temperature and the third temperature is less thanthe first temperature, wherein the temperature of the sample during thesecond time period is caused by heat provided by both the heating deviceand the electromagnetic heating source heating the nucleic acid sample.2. The method of claim 1, wherein the first time period corresponds toan extension phase of a PCR cycle, the second time period corresponds toa denaturation phase of the PCR cycle, and the third time periodcorresponds to an annealing phase of the PCR cycle.
 3. The method ofclaim 1 wherein the heating device is a Peltier device.
 4. The method ofclaim 1 wherein the electromagnetic heating source is an infraredsource.
 5. The method of claim 1 wherein the system further comprises atemperature sensing device and a controller, the controller is operableto control the heating device and the electromagnetic heating source toprovide a desired temperature based on an output of the temperaturesensing device.
 6. The method of claim 5 wherein the temperature sensingdevice is a contact device.
 7. The method of claim 5 wherein thetemperature sensing device is a non-contact device.
 8. The method ofclaim 7, further comprising: switching on a fan to cool the sample fromthe second temperature to the third temperature and switching off thefan when the sample reaches the third temperature.
 9. The method ofclaim 1 further comprising: switching on a fan in communication with themicrofluidic device, thereby increasing the rate of cooling of thesample, and switching off the fan when a desired temperature is reached.10. The method of claim 1, wherein steps (a) through (d) occur while thesample is flowing through a microfluidic channel of the microfluidicdevice.
 11. The method of claim 1, wherein the heating device is incontact with a micro channel containing a nucleic acid sample and theelectromagnetic heating source is remote from the microfluidic device.12. The method of claim 1, wherein the first temperature is about 72°C., the second temperature is about 95° C., and the third temperature isabout 55° C.
 13. The method of claim 1, wherein during the step ofmaintaining the temperature of the sample at about the third temperaturefor a third period of time, the amount of heat provided by the heatingelement is greater than the amount of heat provided by theelectromagnetic heating source.